Chromomycinone glycoside

ABSTRACT

The present invention relates to a new antibiotic-glycoside and to a method of producing the same. The new antibiotic-glycoside has the empirical formula C52H76O24 and comprises aglycone chromomycinone and residues of three desoxy-sugars, two of which are identical to oliose and olivose. The method of producing the antibiotic-glycoside consists in that the culture of Actinomyces olivovariabilis sp.nov., which produces the antibiotic-glycoside and features the following morphological and physiological properties: the sporophores on all the media are spiral-shaped and disposed on short branches either singly, or in clusters; the spirals are contracted and glomerular; the spores are oblong and oval; the envelope is plain; on mineral Hause medium No. 1 the growth is moderate, the colonies are flat, deeply implanted into agar, the aerial mycelium is moderately developed, velvety and dark-grey in colour; the substrate mycelium is olive-grey in colour; soluble pigment is absent; on organic Hause medium No. 2 the growth is moderate, the aerial mycelium is velvety, weakly developed, greyish-violet in colour; the substrate mycelium is dark-olive in colour; the medium is coloured the same hue as the substrate mycelium; on a medium with tyrosine the growth is moderate; the aerial mycelium is weakly developed and is grey in colour; the substrate mycelium is dark-umber in colour; it does not form melamin; does not deliquate gelatine; peptonizes milk by the 20th day; forms H2S; does not hydrolyze starch; does not reduce nitrates; is grown on a nutrient medium comprising the main sources of carbon, nitrogen, and mineral salts; on completion of the growing process, the mycelium is separated, the filtrate of the culture liquid is acidulated to pH 2-4, then the antibioticglycoside is extracted with an organic solvent, the extract is concentrated by evaporation, the desired product is precipitated from the resulting concentrate with an organic solvent, and isolated.

United States Patent [191 Zhdanovich et al.

[ 1 May 13, 1975 CHROMOMYCINONE GLYCOSIDE [76] Inventors: Jury Vasilievich Zhdanovich,

Khoroshevskoe shosse, 5, korpus 8, kv. 21', Galina Borisovna Lokshin, Balaklavsky prospekt, 20, korpus 3, kv. 206; Alexandr Dmitrievich Kuzovkov, ulitsa Vinokurova, 5/6 korpus 2, kv. 33; Sarra Markovna Rudaya, ulitsa Nagornaya, 17, kv. 57; Nadezhda Konstantinovna Solovieva, ulitsa, l8, korpus 2, kv. 151, all of Moscow, USSR.

[22] Filed: Jan. 24,1974

[21] App]. No.: 436,360

Related'U.S. Application Data [63] Continuation of Ser. No. 60917, Aug. 4, 1970,

abandoned.

[30] Foreign Application Priority Data Aug. 4, 1969 U.S.S.R 1355862 [52] U.S. Cl. 260/210 AB; 195/96; 260/210 R;

[51] Int. Cl. C07c 47/18 [58] Field of Search 260/210 AB, 210 R [56] References Cited UNITED STATES PATENTS 3,364,113 l/l968 Friedmann et al 260/210 R Primary Examiner-Johnnie R. Brown Attorney, Agent, or FirmHolman & Stern [57] ABSTRACT The present invention relates to a new antibioticglycoside and to a method of producing the same. The new antibiotic-glycoside has the empirical formula C H O and comprises aglycone chromomycinone and residues of three desoxy-sugars, two of which are identical to oliose and olivose.

The method of producing the antibiotic-glycoside consists in that the culture of Actinomyces olivovariabilis sp.n0v., which produces the antibiotic-glycoside and features the following morphological and physiological properties: the

sporophores on all the media are spiral-shaped and disposed on short branches either singly, or in clusters; the spirals are contracted and glomerular; the spores are oblong and oval; the envelope is plain; on mineral Hause medium No. 1 the growth is moderate, the colonies are flat, deeply implanted into agar, the aerial mycelium is moderately developed, velvety and dark-grey in colour; the substrate mycelium is olive-grey in colour; soluble pigment is absent; on organic Hause medium No. 2 the growth is moderate, the aerial mycelium is velvety, weakly developed, greyish-violet in colour; the substrate mycelium is dark-olive in colour; the medium is coloured the same hue as the substrate mycelium; on a medium with tyrosine the growth is moderate; the aerial mycelium is weakly developed and is grey in colour; the substrate mycelium is dark-umber in colour; it does not form melamin; does not deliquate gelatine; peptonizes milk by the 20th day; forms H 5; does not hydrolyze starch; does not reduce nitrates; is grown on a nutrient medium comprising the main sources of carbon, nitrogen, and mineral salts; on completion of 1 Claim, N0 Drawings CHROMOMYCINONE GLYCOSIDE This is a continuation of application Ser. No. 60,917 filed Aug. 4, 1970, and now abandoned.

The present invention relates to a new antibioticglycoside and to a method of producing the same.

According to the invention, the new antibioticglycoside of the empirical formula C H1 Q comprises aglycone chromomycinone and residues of three desoxy-sugars, two of which are identical to oliose and olivose; the following structural formula is suggested by us for said antibiotic:

OMa

Said antibiotic-glycoside is a pharmacologically active substance and can find application as a medicinal preparation.

According to the supposed mechanism of its action, said antibiotic inhibits DNA (desoxyribonucleic acids)- dependent synthesis of RNA (ribonucleic acids) in various biological systems, such as gram-positive and acidresistant bacteria, in tissue culture, etc.

The new antibiotic-glycoside is a yellow amorphous powder, easily soluble in alcohols and in esters, sparingly soluble in water. Its m.p. is 162-165C (decomp.).

The method of producing the new antibioticglycoside, according to the invention, consists in that the culture of Aczinomyces olivovariabilis sp. nov. which produces the said antibiotic-glycoside and features the following physiological properties: spiral sporophores on all the media that are disposed on short branches either singly or in clusters; the spirals are contracted and glomerular; the spores are oblong and oval, with plain envelopes; on mineral Hause medium No. l the growth is moderate, the colonies are flat, deeply implanted into agar, the aerial mycelium is moderately developed, velvety, dark-grey in colour; the substrate mycelium is 01' ive-grey in colour; soluble pigment is absent; on organic Hause medium No. 2 the growth is moderate, the aerial mycelium is velvety, weakly developed, greyishviolet in colour; the substrate mycelium is dark-olive in colour; the medium is coloured the same hue as the substrate mycelium; on a medium with tyrosine the growth is moderate, the aerial mycelium is weakly developed, grey in colour; the substrate mycelium is darkumber in colour; the culture does not form melanin, does not deliquate gelatine, peptonizes milk by the separated by us and is characterized by the following- 20th day, forms H 8, does not hydrolyze starch, does not reduce nitrates; is grown on a nutrient medium containing the main sources of carbon, nitrogen and mineral salts; on completion of the process of growing, the mycelium is separated, the filtrate of the culture liquid is acidulated to pH=24, after which the antibioticglycoside is extracted with an organic solvent, the extract is concentrated by evaporation, the desired prod uct is precipitated from the resulting concentrate with an organic solvent, and then isolated.

For the process of growing the culture of Actinomyces olivovariabilis sp. nov, it is expedient, that the nutrient medium should have the following composition (the amounts of the constituents being specified in wt.%): soya flour, 2; starch, 2-4; glucose, 2.0; sodium chloride, 0.5; calcium carbonate, 0.5; ammonium sulphate, 0.4; potassium dihydrogen phosphate or potassium hydrogen phosphate, 0.04; water being the balance, the pH of the medium being 6.87.0. The nutrient medium may also have the following composition (in wt.%): corn steep liquor, 1 (green weight); starch, 2.0; glucose, 2.0; sodium chloride, 0.5; calcium carbonate, 0.5; ammonium sulphate, 0.4; water being the balance; the pH of the medium being 6.8-7.0.

For extracting the antibiotic-glycoside from the culture liquid after the mycelium has been separated, ethylacetate or butanol should preferably be used as the organic solvent.

For precipitating the desired product, it is preferable to use diethyl ether or hexane as the organic solvent.

For obtaining a chromatographically pure antibioticglycoside, the isolated desired product is purified by column and thin-layer chromatography methods on silica gel in chloroform-methanol or benzene-acetone systems of solvents.

The present method is effected as follows.

The culture of Actinomyces olivovariabilis sp. nov. is used to produce the new antibiotic-glycoside.

This culture is a species of actinomyces that has been morphological features: the sporophores on all the media employed are spiral-shaped and disposed on short branches either singly or in clusters. The spirals are strongly contacted, have 1-3 turns, and sometimes i are glomerular; the spores are oblong and oval, the envelope is plain.

On mineral House medium No. l the growth is moderate, the colonies are flat, deeply implanted into agar. The aerial mycelium is moderately developed, velvety, dark-grey in colour (ca. A2 according to Bondartsev, W5 by Prauser). The substrate mycelium is olive-grey, dark nut-brown in colour (H1; K5 according to Bondartsev: C6 -C,,, according to Prauser). Soluble pigment is absent. On the Capek medium and on CP of Krasilnikov insignificant differences in the character of growth from the above-cited medium are observed.

On a glucose-asparagine medium the growth is good, the aerial mycelium is velvety, moderately developed, grey in colour. The substrate mycelium is dark-olive, dark-bay in colour (B6, e4 according to Bondartsev). The medium is sometimes coloured the hue of the substrate mycelium.

On a glycerine-asparagine medium the growth is good, the colonies are tuberculate, sometimes fissured. Aerial mycelium is absent. The substrate mycelium is fawn in colour (n17 according to Bondartsev). Soluble pigment is absent.

On a starch-ammonia medium the growth is moderate. The aerial mycelium is velvety, grey in colour. The substrate mycelium is olive-grey, sometimes darkbrown (n colour (M according to Bondartsev, 0.5r according to Prauser). Soluble pigment is absent.

On an oat medium the growth is good. The aerial mycelium is velvety, moderately developed, dark-grey with greenish hue (K5 according to Bondartsev, C6r according to Prauser). The substrate mycelium is darkbay, dark-olive in colour. The medium is slightly coloured the hue of the substrate mycelium; in the course of ageing the medium sometimes acquires pale-crimson colour.

On organic l-lause medium No. 2 the growth is moderate. The aerial mycelium is velvety, weakly developed, greyish-violet or pale-greyish in colour (a3, a6 according to Bondartsev). The substrate mycelium is dark-olive, dark-bay in colour. the medium is coloured the hue of the substrate mycelium.

On a medium with tyrosine the growth is moderate. The aerial mycelium is weakly developed, grey in colour. The substrate mycelium is dark-umber (n2 according to Bondartsev), or dark-bay. Does not form melanin.

The physiological properties of the new species are as follows. It does not deliquate gelatine; peptonizes milk by the th day; forms H 5; does not hydrolyze starch; does not reduce nitrates.

The assimilation of carbon sources on the Preedham- Gottlieb medium is as follows:

arabinose -llrhamnose raffinose sucrose mannitol inositol -ll- In the culture liquid of the actinomyces a pigment of anthocyan type has been found.

In the cource of investigations a great heterogeneity of the culture has been established, said heterogeneity being manifested in a spontaneous formation of a great number of morphological and physiological varieties with persistent traits.

Said culture of Actinomyces olivovariabilis sp. n0v.is seeded onto a nutrient medium and fermented at a temperature of 27-28C for 130-140 hours. Then the resulting culture liquid is filtered off from the mycelium, and the filtrate is acidulated with hydrochloric, sulphuric, or any other mineral acid to pH=24, after which the desired product is extracted with an organic solvent, such as ethylacetate, butylacetate, butanol, etc. It is preferable, that the extraction should be effected with the use of ethylacetate or butanol. For enhancing the quality of the desired product, the extract is treated with an aqueous solution of sodium bicarbon-.

ate, the aqueous phase is acidulated with a minaeral acid (such as hydrochloric, sulphuric, or any other) to pH=2-4, and then the extraction with an organic solvent is repeated.

The resulting extract is concentrated by evaporation to minimum volume, and the desired product is precipitated by using an organic solvent, such as hexane, petroleum ether, diethyl ether, etc. Best results may be obtained when hexane or diethyl ether is used.

For obtaining a chromatographically pure antibioticglycoside, it is additionally purified by column and thinlayer chromatography methodsemployed in succession, on silica gel, in systems of solvents chloroformmethanol (9:1) or benzene-acetone (1:1).

For a better understanding of the present invention, given hereinabelow are examples illustrating the realization of the present method of producing our new antibiotic-glycoside.

EXAMPLE 1 5-10 vol.% of the culture of Actinomyces olivovariabilis sp. nov. are seeded on a nutrient medium of the following composition (in wt.%): soya flour, 2.0; starch, 2.0; glucose, 2.0; sodium chloride, 0.5; calcium carbonate, 0.5; ammonium sulphate, 0.4; potassium dihydrogen phosphate, 0.04; the seeding being effected in an apparatus of lit. capacity. The process of fermentation is run at a temperature of 2628C for hrs.

Then the culture mycelium is separated by filtration, and 40 lit. of the resulting filtrate are acidulated with a 10% hydrochloric acid to pH=22.5 (potentiometrically).

The extraction is effected thrice with ethylacetate (l5, l0 and 7 lit. respectively), the combined ethylacetate extracts are washed with water for removing excess acid (the pH of water washing being about 5), and concentrated by vacuum evaporation to 0.1 of the initial volume. The concentrate is extracted thrice with a 5% aqueous solution of sodium bicarbonate l 0.5 and 0.3 lit. respectively).

The extract is acidulated with a 10% hydrochloric acid to pl-l=22.5 (potentiometrically), and then thrice extracted with ethylacetate (once with 0.4 lit. and two times with 0.2 lit). The combined ethylacetate extract is washed with water, dried over sodium sulphate, concentrated by evaporation, and the antibiotic is precipitated by using a 10 fold volume of hexane.

The precipitate is filtered, washed with hexane, and dried. The yield of the non-purified desired product is 3 g. 0.5 g of the non-purified antibiotic-glycoside, dissolved in a chloroform-methanol mixture (9:1), is introduced into a column (15X700) filled with silica gel which does not contain iron ions.

Development and elution are carried out with the same mixture. The resulting fractions are analyzed by the thin-layer chromatography techniques in the same system of solvents. The fractions that contain the antibiotic-glycoside are combined, vacuum evaporated until dry, dissolved in a small volume of ethylacetate, and applied onto plates with silica gel. After drying, they are chromatographed in the above-described system of solvents.

From the zone with the R, value equal to 0.550.6, by elution with methanol, followed by concentration under a vacuum and reprecipitation from ethylacetate with hexane, the chromatographically pure antibioticglycoside is obtained. The antibiotic glycoside is isolated as a yellow amorphous powder, easily soluble in methanol or ethanol, in esters, and sparingly soluble in" water. the m.p. is l62l65C (decornp).

Calctd., c, 57.76; H, 7.02. c m 0 Found, c, 5790;1-1, 702.

Equivalent weight (as determined by titration with a decinormal solution of caustic soda in a methanolp the process is carried out as described in Example 1.

5 6 benzene medium) is 1073 Calctd. for C l-1 1084. What is claimed is: [041 49+ (with 0.5; ethanol). k 230; l. A glycoside represented by the formula: 280; 317; 330 (inflection), 412 mu (E 174, 380, 59.5, 80). 1/(nujo1 liquid petrolatum, cm) 3400-3450; 1720; 1635; 1580; 1520; 1180; 1070; 1010; 910; 840. 5 (1 3 W EXAMPLE 2 my Up to the step of extracting the antibiotic glycoside,

Extraction is effected thrice with butanol (12, 8 and 10 5 lit.). The combined extracts are washed with a small amount of water and vacuum-evaporated to 0.3 of the initial volume. The concentrate is extracted thrice with a 5% aqueous solution of sodium bicarbonate (1.6, 0.75 and 0.45 lit.). The combined soda extract is acidul5 lated by a 10% hydrochloric acid to pl-1=2-2.5 (potentiometrically), after which it is three times ex- 5 tracted with butanol (once with 0.5 lit. and twice with M1 O o 0.2 lit. each time). The combined butanol extract is. i washed with a small amount of water and evaporated V O until dry.

The residue is dissolved in a small amount of ethylacetate and precipitated with diethyl ether. M

The yield of the antibiotic-glycoside is 2.8 g. H C For obtaining a chromatographically pure antiobiot- 5 ic-glycoside, the product is purified by following the procedure described in Example 1. 

1. A GLYCOSIDE REPRESENTED BY THE FORMULA: 